The goal of this program is to identify, clone, and characterize those genes responsible for the ionizing-radiation resistance of Deinococcus (Micrococcus) radiodurans. This bacterium is the most radiation resistant organism known. Furthermore, its repair of extensive DNA damage of almost all types is error-proof, producing no mutations. Insight into its survival strategies may have broad implications for our understanding of radioresistance. Up until now D. radiodurans has been difficult to approach using standard recombinant-DNA techniques. We have developed a series of new state-of-the-art techniques specifically tailored to D. radiodurans that render this organism wide-open to investigation. Our abilities center on genes that we have identified from other species that express drug-resistance in D. radiodurans, and our demonstrated ability to inert these drug- resistance genes within the D. radiodurans genome at both targeted and random sites and rapidly clone the D. radiodurans sequences flanking these insertions. Capitalizing on these techniques, we will identify those genes necessary for radioresistance and readily clone them by several strategies, each with different tactical advantages. Finally, our capabilities permit easy deliberate construction of double and triple mutants in genes of interest, facilitating genetic pathway analysis of the relevant resistance mechanisms. These studies will lay the genetic groundwork for later investigations into specific enzymatic repair mechanisms employed by D. Radiodurans. This new-found susceptibility of D. radiodurans to molecular genetic analysis should render this remarkably radioresistant organism an important model system for the study of DNA damage tolerance and repair.